HIV infection impairs immunity and immune perturbations persist even after durable virologic control. This population has a high incidence of using prescription opioids or injection opioids that lead to dependence. Opioids are known to be immunosuppressive, and the combination of HIV and opioids can potentially prove to be disastrous for the immune system, yet this area has been grossly understudied. Our recent project on immunity in HIV (AI108472) utilized influenza vaccination as a probe for assessing immunity in HIV+. We observed that HIV+ had lower vaccine responses than uninfected, and a subgroup of HIV+ drug abusers had worse antibody response than non-abusers. We could classify participants as vaccine responders (VR) and vaccine non-responders (VNR). In studying mechanisms of immune defects in VNR, we identified quantitative and qualitative defects in peripheral T follicular helper cells (pTfh), which are a subset of CD4 T cells that are essential for vaccine-induced antibody responses. The pTfh displayed a skewed polarization away from a favorable IL-21 secreting phenotype towards a detrimental IL-2 secreting Th1 phenotype, coupled with abundance of inflammatory markers, resulting in failure of pTfh to provide B cells with the helper signals required for secreting antibodies. Our central hypothesis is that chronic opioid use blunts the immune response to impair the generation of antibodies and exacerbates the immune dysfunction of HIV even in virally controlled patients. We will recruit opioid users and non- users in HIV negative and virally suppressed HIV+ populations to address key questions on the effect of opioids on the antibody response following seasonal influenza vaccination. The project has 3 aims: 1. To evaluate basal state of inflammation and immune/activation in relation to pTfh phenotype and the magnitude and quality of flu vaccine response. 2. To characterize pTfh by phenotypic, functional and transcriptomic analyses and 3. To test interventions for potential reversal of opioid induced immune deficiency in VNR. We will use a combination of single cell technologies to gain high resolution datasets including multi-parameter flow cytometry and the 10x single cell genomics platform combined with indexed surface marker expression using novel nucleic acid-tagged antibodies. In order to gain a comprehensive understanding on the topic of opioid effect on immune function in presence of HIV infection, we will evaluate immune cells ex vivo from the populations of interest using unbiased genomics approaches to obtain a snapshot of immune perturbation compared to healthy controls. In vitro studies with purified cell subsets and addition of morphine during antigen exposure will allow for mechanistic evaluation of opioid impact on the immune system. These studies are feasible, given our expertise in the technologies described and access to the required population through our ID Clinics and specialized programs dealing with opioid addicted populations. We expect to provide novel insight into immune perturbations that will help in strategizing vaccine approaches, including those for opioid vaccines.